1. Field of the Invention
This invention relates to novel multibinding compounds (agents) that are useful as antibiotics, and to pharmaceutical compositions comprising such compounds. The compounds are useful medications for the prophylaxis and treatment of various bacterial infections.
The following publications are cited in this application:
Storm et al., xe2x80x9cPolymyxin and related peptide antibiotics,xe2x80x9d Ann. Rev. Biochem., 46: 723-763 (1977).
Weinstein et al., xe2x80x9cSelective chemical modifications of Polymyxin B,xe2x80x9d Bioorganic and Medicinal Chemistry Letters, 8: 3391-3396 (1998).
Kimura and Matsunaga, xe2x80x9cPolymyxin B octapeptin and polymyxin B heptapeptide are potent outer membrane permeability-increasing agents,xe2x80x9d Journal of Antibiotics, 45(5): 742-749 (1992).
Srinivasa and Ramachandran, xe2x80x9cChemical modification of peptide antibiotics: Part VI-Biological activity of derivatives of polymyxin B,xe2x80x9d Indian J. Biochemistry and Biophysics, 14: 54-58 (1978).
PCT WO 88/00950 to Fauchere et al.
DE Patent No. 1,906,699 to Pfizer.
DE Patent No. 2,204,887 to Rhone-Poulenc.
J. E. Kapusnik-Uner, M. A. Sande, H. F. Chambers in Goodman and Gilman""s xe2x80x9cThe Pharmacological Basis of Therapeutics,xe2x80x9d 9h Ed. (J. G. Hardman, L. E. Limbird, P. B. Molinoff, R. W. Ruddon, A. G. Gilman, Eds.); McGraw-1-Ell, New York: p 1123-1153 (1996).
M. R. W. Brown, S. M Wood, J. Pharm. Pharmacol. 24: 215-218 (1972).
S. Srimal, N. Surolia, S. Balasubramanian, A. Surolia, Biochem. J., 315: 679-686 (1996).
J. L. Shenep, R. P. Barton, et al., J. Infect. Dis. 151: 1012-1018 (1984).
M. G. Tauber, A. M. Shibl, C. J. Hackbarth, J. W. Larrick, M. A. Sande, Antimicrob. Agents Chemother. 156: 456-462 (1987).
G. S. Doig, C. M. Martin, et al., Crit. Care Med. 25:1956-1961 (1997).
C. Verwaest, J. Verhaegen, P. Ferdinande, M. Schetz, G. Van Den Berghe, L. Verbist, P. Lauwers, Crit. Care Med. 25: 63-71 (1997).
G. S. Bauldoff, D. R. Nunley, J. D. Manzetti, J. H. Dauber, R. J. Keenan, Transplantation 64: 748-752 (1997).
P. Diot, F. Gangadoux, C. Martin, H. Ellataoui, Y. Furet, M. Breteau, E. Boissinot, E. Lemarie, Eur. Resp. J. 10: 1995-1998 (1997).
S. E. Bucklin, P. Lake, L. Logdberg, D. C. Morrison, Antimicrobial Agents Chemother. 39: 1462-1466 (1995).
B. L. Jaber, B. J. Pereira, Am. J. Kidney Dis. 30(Suppl. 4): S44-56 (1997).
J. R. Berg, C. M. Spilker, S. A. Lewis, J. Membrane Biol. 154: 119-130 (1996).
L. Weinstein in xe2x80x9cThe Pharmacological Basis of Therapeutics,xe2x80x9d 5 1h Ed. (L. S. Goodman, A. Gilman, Eds.); MacMillan, N.Y.:, p1230-1233 (1975).
Physician""s Desk Reference, Medical Economics Co., Oradell, N.J.: (1993).
M. Helander, Y. Kato, I. Kilpelainen, R. Kostiainen, B. Lindner, K. Nummila, T. Sugiyama, T. Yokochi, Eur. J. Biochem. 237: 272-278 (1996).
Entries 2542 and 7734, The Merck Index, 12 th Edition, Merck and Co., Whitehouse Station, N.J.: (1996).
K. Vogler, R. O. Studer, W. Lergier, P. Lanz, Helv. Chim. Acta 43: 1751-1760 (1960).
K. Vogler, R. O. Studer, P. Lanz, W. Lergier, E. Boehni, Experientia 20: 365-366 (1964).
T. Kurihara, H. Takeda, H. Ito, Yakugaku Zasshi 92: 129-134 (1972).
K. Nakajima, Chem. Pharm. Bull. 15: 1219-1224 (1967).
M. Teuber, Z. Naturforsch. Teil B 25: 117 (1970).
Y. Kimura, H. Matsunaga, M. Vaara, J. Antibiot. 45: 742-749 (1992).
S. Chihara, A. Ito, M Yahata, T. Tobita, Y. Koyama, Agric. Biol. Chem. 38: 521-529 (1974).
S. Chihara, A. Ito, M Yahata, T. Tobita, Y. Koyama, Agric. Biol. Chem. 38: 1767-1777 (1974).
S. Chihara, T. Tobita, et al., Agric. Biol. Chem. 37: 2455-2462 (1973).
D. A. Stoma, K. S. Rosenthal, P. E. Swanson, Ann. Rev. Biochem. 46: 723-763 (1977).
M. Vaara, Drugs Exp. Clin. Res. 17: 437-444 (1991).
PCT WO 90/15628.
G. Radhakrishna, L. K. Ramachandran, Indian J. Biochem. Biophys. 20: 213-217 (1983).
C. P. Coyne, J. T. Moritz, J. Endotoxin Res. 1: 207-215 (1994).
J. L. Fauchere, K. Mosbach CH Appl. 86/315106 August 1986.
All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
2. State of the Art
Bacteria are ubiquitous microbes capable of causing significant morbidity and mortality in infected individuals. Healthy individuals, having intact immune systems, rapidly eliminate pathogenic bacteria. However, many conditions render patients vulnerable to bacterial infection. Thus, individuals suffering from primary immunodeficiency disorders, such as AIDS, commonly develop infections. Alternatively, individuals may become susceptible to bacterial infection as the result of secondary immunodeficiencies due to other underlying disorders. For example, patients with diseases such as diabetes, connective tissue disorders, or trauma frequently develop complications due to severe bacterial infections. In such patients, overwhelming bacterial infections may result in a cascade of physiological changes leading to septic (or endotoxic) shock, which often culminates in the patient mortality.
Although most bacteria are capable of producing sepsis, a sub-class of bacteria, known as Gram-negative bacteria, which includes Eschericia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa are the usual etiologic agents. The profound pathogenic effects of these microbes are due to a structural component, unique to Gram-negative bacteria, known as an outer membrane.
The outer membrane, which surrounds the bacterial cell, and protects it from environmental assaults, includes a molecule known as lipopolysaccharide (LPS). LPS is a complex structure with three components: 1) an outer region consisting of polymerized di- to penta-saccharide repeating units whose composition varies with bacterial species; 2) an inner region including of oligosaccharides linked by a sugar 2-keto-3-deoxy-C-mannose-octonate to a disaccharide backbone; and lipid A. This latter molecule, a glucosamine disaccharide with attached phosphate and acyl (fatty acid) groups, is responsible for most of the biological activity of the molecule.
The pathological effects of LPS are due to both intact LPS present in the outer membrane of the cell (bound LPS) and LPS that is released from the membrane and shed into blood (soluble LPS). Regardless of form, LPS elicits its biological effects by binding to a receptor found on a mononuclear phagocyte known as a monocyte. The interaction stimulates cellular processes resulting in the release of pro-inflammatory mediators such as TNF-xcex1, IL-1xcex2, IL-6 and PGE2, which, then leads to arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea and organ dysfunction that characterize septic shock.
Bacterial infections are usually treated with a molecularly diverse group of agents known as antibiotics, which act by a wide variety of mechanisms well known to those skilled in the art. While these drugs are sometimes capable of resolving the effects of bacteremia, infections with Gram-negative bacteremia presents special challenges. For example, treatment with conventional antibiotics while leading to the death of the pathogen, results in the release of toxic bacterial components, such as LPS. Thus, treatment with antibiotics may increase the amount of LPS or products of LPS such as endotoxin into the circulation.
Certain antibiotics, however, are able to neutralize the action of LPS, and mitigate its effects by binding to the molecule. Examples of such antibiotics include the polymyxin, circulin and octapeptin antibiotics, most notably polymyxin B and polymyxin E (also known as colistin), which are cyclic polypeptide compounds produced by strains of Bacillus polymyxa. 
The lipid-bearing, polycationic polymyxin forms a complex with anionic phospholipids of the LPS and inserts into the membrane. This event disrupts the LPS, leading to loss of essential intracellular components and rapid bacterial cell death. In addition to its killing effects on intact bacteria, polymyxin also has high affinity for xe2x80x9cfreexe2x80x9d LPS components, most importantly, the lipid A portion of the lipopolysaccharide of the LPS (endotoxin). Complexation of lipid A by polymyxin prevents most of the pathophysiologic consequences of endotoxin in experimental systems.
Combinations of polymyxin B sulfate and/or colistin sulfate with various other compounds are widely used in opthalmic, otic, and topical applications against Gram-negative organisms. Strains of Enterobacter, E. coli, Klebsiella, Salmonella, Pasteurella, Bordetella, and Shigella are typically sensitive to polymyxins at concentrations of 0.05-2.0 micrograms/mL in vitro, while most strains of Pseudomonas aeruginosa are inhibited by less than 8 micrograms/mL. Intrinsically resistant strains include Proteus mirabilis, Serratia marcesens, Providencia, and Edwardsiella tarda. More recently introduced uses of the polymyxins include the use of oral colistin for prophylactic gut clearance and of nebulized colistin for treatment of Pseudomonas infections in cystic fibrosis patients. In current development are the systemic use of macromolecular polymyxin-dextran conjugates and the extracorporeal use of polymyxin adsorbents for intervention in Gram-negative sepsis.
Polymyxin was at an earlier time administered parenterally and colistin sulfate is formulated for parenteral use; however, parenteral use of polymyxins is rare due to their nephrotoxicity and neurotoxicity. These effects are believed to have as their source the interaction of the polymyxins with phospholipids of mammalian cells. Neurological side effects of colistin administration include circumoral parasthesia pain at the site of intramuscular injection, numbness, tingling, or formication in the extremities, generalized pruritis, vertigo, dizziness, slurring of speech, and respiratory paralysis via neuromuscular blockade. Nephrological side effects include acute tubular necrosis, interstitial nephritis, proteinuria, hematuria, cylindruria, azotemia, and reduced glomerular filtration rate. The magnitude of these effects increases with continued therapy; however, the effects are generally reversible upon cessation of treatment.
For these reasons, polymyxin B, in intravenous form, is only given to hospitalized patients under constant supervision. Polymyxins and related antibiotics are not used routinely for systemic infections. Application of polymyxins to intact skin, denuded skin, or mucous membranes results in no systemic reactions because the drugs are poorly absorbed. Side effects following large (600 mg) oral doses of the antibiotic include nausea, vomiting, and diarrhea. Neurotoxic reactions have additionally been observed, the most severe being respiratory paralysis when given soon after anesthesia and/or muscle relaxants.
Given polymyxin""s significant systemic toxicities, the advent of anti-pseudomonal xcex2-lactams, the fluoroquinolone family of antibacterial agents, and aminoglycosides effective against Gram-negative organisms superseded parenteral use of polymyxin.
Antibacterial agents are important weapons in the fight against pathogenic bacteria. However, an increasing problem with respect to the effectiveness of antibacterial agents relates to the emergence of strains of bacteria that are highly resistant to such agents. Microbial resistance to the polymyxins is slow to develop and typically involves alterations in the composition of LPS components.
It would therefore be desirable to find antibacterial agents that are active against Gram-negative bacteria, in particular, drug resistant strains. It would also be advantageous to have antibacterial agents that demonstrate high activity and selectivity toward their targets and have high bioavailability, low toxicity, for example, nephrotoxicity, and other side effects. The present invention provides such agents.
This invention is directed to novel multibinding polymyxin, circulin and octapeptin antibiotics. The multibinding compounds of this invention are useful in the treatment and prevention of bacterial infections.
In particular, the compounds can be used to treat bacterial infections caused by Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, Enterobacter sp., Salmonella sp., Pasteurella sp., Bordetella sp., Shigella sp., Proteus mirabilis, and Serratia marcesens. 
In one of its composition aspects, this invention provides a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a polymyxin, circulin or octapeptin antibiotic or other suitable compound which binds to the LPS present in bacteria, in particular, Gram-negative bacteria.
In another of its composition aspects, this invention provides a multibinding compound of formula I:
(L)p(X)qxe2x80x83xe2x80x83I
wherein each L is independently a ligand comprising a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS and/or which demonstrates antibacterial properties; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; and pharmaceutically-acceptable salts thereof.
Preferably, q is less than p in the multibinding compounds of this invention.
Examples of suitable ligands include polymyxin A, polymyxin B1, polymyxin B2, polymyxin D1, polymyxin E1, polymyxin E2, circulin A, octapeptin A1, octapeptin A2, octapeptin A3, octapeptin B1, octapeptin B2, octapeptin B3, octapeptin C1. The xcex3-amino group on the DAB subunits on the ligands is a preferred site for attachment to the linker(s).
In still another of its composition aspects, this invention provides a multibinding compound of formula II:
Lxe2x80x2xe2x80x94Xxe2x80x2xe2x80x94Lxe2x80x2xe2x80x83xe2x80x83II
wherein each Lxe2x80x2 is independently a ligand comprising a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS and/or which demonstrates antibacterial properties; and Xxe2x80x2 is a linker; and pharmaceutically-acceptable salts thereof.
Preferably, in the above embodiments, each linker (i.e., X, Xxe2x80x2 or Xxe2x80x3) independently has the formula:
xe2x80x94Xaxe2x80x94Zxe2x80x94(Yaxe2x80x94Z)mxe2x80x94Ybxe2x80x94Zxe2x80x94Xaxe2x80x94
wherein
m is an integer of from 0 to 20;
Xa at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)NRxe2x80x94, xe2x80x94C(S), xe2x80x94C(S)Oxe2x80x94, xe2x80x94C(S)NRxe2x80x94 or a covalent bond where R is as defined below;
Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
Ya and Yb at each separate occurrence are selected from the group consisting of xe2x80x94C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(O)xe2x80x94, xe2x80x94NRxe2x80x2C(O)NRxe2x80x2xe2x80x94, xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90C(Xa)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94P(O)(ORxe2x80x2)xe2x80x94Oxe2x80x94, xe2x80x94S(O)nCRxe2x80x2Rxe2x80x3xe2x80x94, xe2x80x94S(O)nxe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94 and a covalent bond; where n is 0, 1 or 2; and R, Rxe2x80x2 and Rxe2x80x3 at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic.
In yet another of its composition aspects, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrates antibacterial properties; and pharmaceutically-acceptable salts thereof.
This invention is also directed to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of formula I or II.
The multibinding compounds of this invention are effective antibiotics, which are useful for treating a variety of bacterial infections. Accordingly, in one of its method aspects, this invention provides a method for treating various bacterial infections. Examples of such infections include infections caused by Enterobacter sp., E. coli, Klebsiella sp., Salmonella sp., Pasteurella sp., Bordetella sp., Shigella sp., Pseudomonas aeruginosa, Proteus mirabilis, and Serratia marcesens. 
When used to treat bacterial infections, for example, the method involves administering to a patient having a bacterial infection a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrates antibacterial properties; and pharmaceutically-acceptable salts thereof.
This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to various sites on bacteria. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarizability and/or polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to sites on various bacteria. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarizability and/or polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
This invention is also directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to various sites on bacteria. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands which bind to the LPS present in bacteria, especially gram negative bacteria, and/or which demonstrate antibacterial properties.
Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties, which method comprises:
(a) identifying a ligand or a mixture of ligands which includes a polymyxin, circulin or octapeptin compound or other suitable compound which binds to the LPS present in bacteria and/or which demonstrates antibacterial properties, wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.
In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties, which method comprises:
(a) identifying a library of ligands which includes polymyxin, circulin or octapeptin and other suitable compounds which bind to the LPS present in bacteria and/or which demonstrate antibacterial properties wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.
The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b). Sequential addition is preferred when a mixture of different ligands is employed to ensure heteromeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer compounds prepared are homomultimeric compounds.
The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).
In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties which library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands which include polymyxin, circulin or octapeptin compounds or other suitable compounds which bind to the LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrate antibacterial properties; wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties and which may possess multivalent properties which library is prepared by the method comprising:
(a) identifying a library of ligands which includes a polymyxin, circulin or octapeptin compound or other suitable compounds which bind to the LPS present in bacteria and/or which demonstrate antibacterial properties wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarizability and/or polarization and amphiphilic linkers. For example, in one embodiment, each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100 xc3x85.
In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on the ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
In other embodiments, the multimeric ligand compound is homomeric (i.e., each of the ligands is the same, although it may be attached at different points) or heteromeric (i.e., at least one of the ligands is different from the other ligands).
In addition to the combinatorial methods described herein, this invention provides for an iterative process for rationally evaluating what molecular constraints impart multibinding properties to a class of antibacterial multimeric compounds or ligands. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties with respect to the LPS present in certain bacteria, especially Gram-negative bacteria, or which possess antibacterial properties, which method comprises:
(a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which bind to the LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrate antibacterial properties; with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties;
(c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties;
(d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)-(c) above;
(e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration;
(f) evaluating what molecular constraints imparted enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above;
(g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.
Preferably, steps (e) and (f) are repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.